Recombinant DNA technology provides a valuable means of synthesizing large amounts of desirable eukaryotic proteins such as mammalian hormones, interferons, and enzymes. While organisms can be readily manipulated in order to produce the desired protein, the host organism does not normally secrete the protein product into the culture medium. Thus lysis of the organisms, e.g., bacteria, followed by isolation of the desired protein is usually necessary.
Generally, the first step in the purification of native and recombinant proteins entails lysis of the cells producing the proteins, resulting in liberation of the cellular components. Classic physical methods for cell lysis include sonication and the use of a French Pressure Cell, often in combination with a chemical or enzyme agent to aid in lysis. Lysis by physical methods produces membrane fragments and small DNA molecules caused by shearing of the chromosomal DNA, either of which can interfere with subsequent separation and/or analysis of the desired proteins. Removal of these contaminants requires additional costly and time-consuming purification steps, including DNA digestion.
Classic protein purification methods include precipitation (e.g. PEI, PEG, and ammonium sulfate), filtration, preparative electrophoresis and the like. These methods are often performed on bacterial lysates or partially purified preparations of protein. Additional methods based on chromatography include, but are not limited to, ion-exchange chromatography, size-exclusion chromatography, hydrophobic interaction chromatography, and affinity chromatography. Any and all of these methods are dependent on an efficient lysis procedure in order to insure adequate yield.
While methods exist in the art for lysis of cells, there exists a need in the art for a rapid method which employs a gentle cell lysis and avoids mechanical disruption; separation of the protein and peptide of interest from contaminating cellular debris, including DNA and membrane fragments; and additional purification methods into one or a few procedures. The present invention provides such compositions, methods and kits.